Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle

Abstract

The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates.

Figure 1

The ClpXP protease. a) Side view of ClpXP degrading a substrate (green). A ClpX hexamer (blue) recognizes, unfolds, and translocates protein substrates into the degradation chamber of ClpP (dark orange), which consists of two heptameric rings. ClpXP is principally stabilized by interactions between the IGF loops of ClpX and hydrophobic clefts on each ClpP ring. b) Axial view of a ClpX homohexameric ring and a ClpP homoheptameric ring, highlighting the interaction elements. c) Chemical structure of ADEP-2B.^, The portion thought to mimic binding of an IGF tripeptide is colored purple. d) ^sc6ClpX^ΔN-bio is a single-chain pseudohexamer in which the ClpX^ΔN subunits are linked by six-residue tethers. The protein contains an N-terminal FLAG tag and a C-terminal sequence consisting of a biotin acceptor peptide (BAP), a cleavage site for Tobacco Etch Virus protease (TEV), and six histidines (H₆).

Figure 2

Association of ClpP with ^sc6ClpX^ΔN-bio assayed by BLI. a) A streptavidin-coated BLI biosensor was incubated sequentially with buffer, buffer plus 20 nM ^sc6ClpX^ΔN-bio, buffer plus 2 mM ATP, buffer plus 200 nM ClpP and 2 mM ATP, and buffer plus 2 mM ATP. b) BLI trajectories showing that ClpP binding to ^sc6ClpX^ΔN-bio occurs with similar kinetics in the presence of ATP or ATPγS (2 mM each). Binding was not observed with 2 mM ADP or no nucleotide. Individual trajectories are offset to allow comparisons. c) Residuals of single-exponential and/or double-exponential fits for association trajectories obtained using ClpP concentrations of 0.5 or 500 nM. d) For ClpP concentrations of 200 nM or less, rate constants from single-exponential fits of ClpP association trajectories (k_obs) varied linearly with ClpP, with a slope corresponding to the second-order association rate constant. e) Variation of the rate constants from double-exponential fits for ClpP concentrations of 500 nM or higher. The curves are fits to a hyperbolic equation. For k_obs-1 (amplitude ~70%), the maximal rate was 22 ± 7 s⁻¹ with a half-maximal concentration of ~35 μM ClpP heptamer. For k_obs-2 (amplitude ~30%), the maximal rate was 0.54 ± 0.2 s⁻¹ with a half-maximal concentration of ~2 μM ClpP heptamer.

Figure 3

Nucleotide dependence of ClpP association. a) Graphs showing normalized ^sc6ClpX^ΔN-bio ATP-hydrolysis activity and normalized ClpP association rate constants (obtained using 200 nM ClpP) as a function of ATP concentration. The curves are fits to a Hill equation (Y = [ATP]ⁿ/(K_appⁿ + [ATP]ⁿ). For ATP hydrolysis, the fitted values of K_app and n were 42 ± 3 μM and 2.4 ± 0.3, respectively. For assembly, these fitted values were 100 ± 2 μM and 2.1 ± 0.06, respectively. Maximal fitted values prior to normalization were 73 ± 1 min⁻¹ enz⁻¹ for ATP hydrolysis and 0.138 ± 0.001 s⁻¹ for k_obs. b) Variation of k_obs for 200 nM ClpP association with the fraction of ATP in mixtures with ADP (2 mM total nucleotide).

Figure 4

Dissociation and equilibrium stability of ClpXP complexes. a) Complexes were assembled with ^sc6ClpX^ΔN-bio bound to the biosensor, 200 nM ClpP, and 2 mM ATP. At time zero, the biosensor was moved into ClpP-free buffer containing 2 mM ADP, 2 mM ATP, or 2 mM ATPγS. The trajectories have been offset vertically, but all start at the same BLI value ± 5%. b) The same experiment as shown in panel A, except ClpXP complexes were assembled in the presence of 2 mM ATPγS. c) Dissociation kinetics after transfer into buffer without nucleotide for ClpP complexes assembled with ATPase-active ^sc6ClpX^ΔN-bio and ATP (bottom curve), ATPase-active ^sc6ClpX^ΔN-bio and ATPγS (middle curve) or a ATP-hydrolysis defective REEREE ^sc6ClpX^ΔN-bio variant and ATP (top curve). d) Complexes were assembled with ATP as in panel a and then transferred into ClpP-free buffer containing different concentrations of ATP. Dissociation rate constants were calculated from single-exponential fits and plotted as a function of the ATP concentration. The line is a fit to the Hill equation. e) Half-lives of ClpXP complexes assembled in ATP following transfer into buffer containing 100% ADP (2 mM) or 87.5% ADP (1.75 mM ADP; 0.25 mM ATP). (f) BLI trajectories showing that ClpXP complexes are stable for long periods in ClpP-free buffer containing 2 mM ATP, 2 mM ATP and unbiotinylated ^sc6ClpX^ΔN (1 μM), or 2 mM ATP and GFP-ssrA (20 μM). g) Equilibrium BLI response for ^sc6ClpX^ΔN-bio binding as a function of total ClpP concentration. The fitted curve is a hyperbolic equation with half-maximal binding at a total ClpP concentration of 160 ± 75 pM.

Figure 5

ADEP effects. a) BLI trajectories following transfer of ATP-stabilized ClpXP into ClpP-free buffer containing 2 mM ATP without or with 50 μM ADEP-2B. b) BLI trajectories following transfer of ATP-stabilized ClpXP into ClpP-free buffer containing 200 nM ADEP-2B and 2 mM ATP or ATPγS. c) ADEP-2B stimulation of dissociation. ATP-stabilized ClpXP was transferred into ClpP-free buffer containing 2 mM ATP and different ADEP-2B concentrations, and dissociation rate constants were determined by single-exponential fits. The line is a hyperbolic fit (R² = 0.994) with values of 2.0 ± 0.13 s⁻¹ for the maximum rate and 50 ± 7 μM for half-maximal stimulation. Sigmoidal equations for mechanisms involving two ADEPs (α²/(1+2α+α²); α = [ADEP]/K_μ; R² = 0.971) or three ADEPs (α³/(1+3α+3α²+α³); R² = 0.958) gave poorer fits. d) (top) ADEP-2B stimulation of decapeptide cleavage of 25 nM ClpP. The line is a hyperbolic fit (R² = 0.995) with half-maximal inhibition at a total concentration of 240 ± 26 nM. (bottom) ADEP-2B inhibition of association of 200 nM ClpP. The line is a hyperbolic fit (R² = 0.981) with half-maximal inhibition at a total concentration of 167 ± 20 nM.

Figure 6

Models for small-molecule control of complex stability. a) ADEP binding to an empty ClpP cleft allosterically stabilizes a conformation from which ClpX rapidly dissociates. For simplicity, only a subset of IGF loops in ClpX and clefts in each ring of ClpP are shown. b) ADEP binding to a ClpP cleft transiently unoccupied by an IGF loop prevents re-docking and stimulates dissociation. c) ATP binding to the ClpX hexamer stabilizes a conformation of the IGF loops that binds the ClpP clefts more efficiently. d) ATP binding to the ClpX hexamer positions the IGF loops to interact optimally with ClpP.